Method for the filtration of virus fluids



30,1952 w. AUERSWALD ETT'AL 3,

METHOD FOR THE FILTRATION OF VIRUS FLUIDS Filed July 10, 1959 INVENTORS.Hflwerswald United tates Patent 3,061,518 METHOD FOR THE FILTRATION 0FVIRUS FLUIDS Wilhelm Auerswald, 22 Wahringer Strasse, Vienna IX,

Austria, and Johann Eibl, 1 Wohllebeugasse, Vienna IV, Austria FiledJuly 10, 1959, Ser. No. 826,268 Claims priority, application AustriaJuly 2, 1959 5 Claims. (Cl. 16778) This invention relates to a methodfor the filtration of virus fluids; in particular the invention relatesto the elimination of the antigen-adsorption activity of pore filtersdestined for the filtration of virus fluids.

The filtration of virus suspensions is a crucial step in processingvirus suspensions for vaccine production. For instance in the productionof poliomyelitis vaccine by infecting tissue cultures with the virus,multiplication of the virus occurs and finally the culture fiuidcontains high concentrations of virus. In order to get this virussuspension free from bacteria, aggregates and minute parts of destructedcells, filtration through pore filters, sc-called bacteria tight filtersis necessary (Regulations of Biological Products, revised August 1956,U.S. Department of Health, Education and Welfare 73 100-73 105). Such afiltration, however, always involves a significant loss of antigenicitythat can be demonstrated by titration according to the tissue culturetechnique. This means that while obtaining a filtrate which is free fromconcomitant bacteria and aggregates an undesirable loss of the antigenicvalue of the virus suspension occurs. The reason for these losses duringfiltration is the high adsorptive capacity of the inner surfaces of thefilter pads or pores.

Several attempts have been made to decrease the loss of antigenicityduring filtration. First of all, attention has been given to the filtermaterials and it has been found that filter pads of the cellulose type,such as Schleicher and Schuell membrane filters, are suitable, whereasasbestos filters must be excluded from virus filtration due to theirextraordinary high adsorptive power. Furthermore it has been proposed topretreat the filter pads before filtering the virus suspension withgelatine or high polypeptides. But these materials whose appearance inthe final vaccine cannot be fully avoided, involve certain risks for thevaccinees. According to the common vaccination schedules, virus vaccinesmust be injected parenterally repeatedly. The materials mentionedhereinbefore may sensitize a vaccinee and induce allergic reactions.

The main object of the present invention is to avoid these diificultiesand to provide a safe virus vaccine free from bacteria, aggregates andother undesirable contents and having a high level of antigenicity.

A further object of the invention is to eliminate the loss ofantigenicity in a given virus suspension during filtration by saturatingthe adsorptive capacity of the filter pores or of the active innersurf-aces of the filter pads.

According to the invention a pore filter destined for the filtration ofvirus fluids is treated with a human protein solution which had beenpreviously subjected to an inactivation procedure in order to avoid anyrisk of transmitting active virus of the homologous hepatitis serum intothe filtrate by said human protein solution. The inactivated proteinsolution from human origin must also be free from denatured proteinfractions which could act as new antigens, and must not contain proteinswhich could give virus neutralizing effects. It has been found that allrequirements are optionally fulfilled when proteins from human originare used having an electrophoretic mobility of the classes between -8.0and -3.() cm? voltsec. and being stable during a 10 hours heat treatmentat a temperature of about 60 C. after which treatment their turbidity isnot more than 30 Mueller units.

It is understood by one skilled in the art that the determination of theelectrophoretic mobility is effected in a barbiturate buffer solution ata pH value of 8.6 and the turbidity is measured in a 5 percent solution.According to the definition of a Mueller unit, one unit measured in theMueller nephelometer at 5461 Angstrom units corre sponds to 0.0010 cm.-absolute turbidity.

Examples of human proteins which may successfully be used in the processaccording to the invention are the P.P.F. human plasma protein fractionand the human albumin. The P.P.F. human plasma protein fraction is thatproduced in accordance with the provisional minimum requirements of theU.S. Department of Health,

Education and Welfare, Public Health Service, National Institutes ofHealth, Bethesda 14, Maryland, of August 14, 1958, certain of which areas follows:

(1) Proper name and aefiniti0n.The proper name of this product shall bePlasma Protein Fraction (Human) and it is defined as a product derivedfrom human blood consisting of not less than :2.7 globulins other thangamma globulin by moving boundary electrophoresis.

(2) S0urce.--Plasma Protein Fraction (Human) is made from hum-an plasmacontaining not more than 25 mg. of hemoglobin per ml. Donors of theplasma shall be in good health and free of disease transmissible by heattreated blood fractions as determined by medical history and suchphysical examination as appears necessary at the time of bloodcollection. No individual shall be used as a source of Plasma ProteinFraction (Human) if he has (a) A history of viral hepatitis;

(11) A history of close contact within six months of donation with anindividual having viral hepatitis;

(0) A history of having received within six months human blood, or anyderivative of human blood which the Division of Biologics Standards hasadvised the licensed establishment is a possible source of hepatitis. A

(3) Processing meth0d.-The processing method shall be one that uniformlyyields a protein mixture which does not show more than a 5% apparentincrease in alpha globulin (T-globulin) after heating at 60 for tenhours; which contains less than 1.7 gamma globulin as measured by theelectrophoretic method described herein and which contains less than 5%of protein with a sedimentation constant of over 7.0. 'The processingmethod shall yield a product which is safe and suitable for intravenousinjec tion. The various steps in processing shall be conducted in asaseptic a manner as possible and every precaution of technique andcleanliness of equipment must be ob served to prevent unnecessarycontamination either with bacteria or other deleteriousmatter-particularly pyrogenic material. A preservative 0r bacteriostaticagent shall not be used toprevent bacterial growth during procf essmg.

(4) Other processing.As soon as possible after removing theothercomponents of plasma from Plasma Protein Fraction, makingelectrolyte and pH adjustments,'and adding the stabilizer, it shall befiltered to sterilize and remove particulate matter. The entire contentsof each bulk container shall befilled into final containers during asingle filling operation. If issued as a liquid the prod uct shall beheated in the final container at an attained: temperature of 60 C. fornot less than 10 hours and all final containers shall be held at 18 to35 C. for'not less than 7 days and examined for visible evidence ofbacterial growth. If issued as a dried product, Plasma Protein Fraction(Human) shall be heated as above before drying;

3 (5) Control tests on the product.Each lot of Plasma Protein Fraction(human) shall pass the following tests:

(a) Sterility.-Plasma Protein Fraction (Human) in final containers shallpass the tests for sterility.

Stability test.-The turbidity is measured by comparison of the light ofwave length 5461 A. (from a General Electric H4 mercury vapor lampthrough a Corning No. 3486 filter) scattered at an angle of 45 and thattransmitted at One unit of the Mueller nephelometer as currently usedcorresponds to 0.0010-1 absolute turbidity. Plasma Protein Fraction(Human) shall meet the following criteria:

(1) The turbidity of the 5% Plasma Protein Fraction (Human) solutionafter heat treatment must not be more than 30 units. (2) The turbiditymust not increase more than units when the sample is heated for 50 hoursat 57 C. and after agitation in a mechanical shaker for 6 hours.

(0) The final product, if a liquid, shall contain 5i0.25% of proteinfrom human plasma and if dried shall be accompanied by diluent toreconstitute to this protein concentration.

Safety test.-Plasma Protein Fraction (Human) shall pass the test forsafety.

(e) Pyrogen test.--Plasma Protein Fraction (Human) shall not cause atemperature rise of 1.1 C. or more in rabbits when injected in an amountof 3.0 ml. per kilo of rabbit weight.

(f) Sodium and potassium content.-The sodium concentration shall notexceed 0.15 $0.01 M. The potassium concentration shall not exceed 0.001M. The flame photometer is considered adequate for these tests.

(g) pH .--The pH of the final product is 7:0.3 as measured in a solutiondiluted to 1 percent protein in 0.15 M. NaC

The human albumin is that produced in accordance with the minimumrequirements for human albumin of the US. Department of Health,Education, and Welfare, Public- Health Service, National Institutes ofHealth, Bethesda 14, Maryland, 8th revision, July 30, 1953, certain ofwhich are as follows.

Normal Serum Albumin (Human):

1. The product- 1.1 Proper name.The proper name is Normal Serum Albumin(Human).

1.2 Definiti0n.Normal Serum Albumin (Human) is a sterile preparation ofthe serum albumin component of human blood.

1.3 Source-Normal Serum Albumin (Human) is prepared from human bloodcollected in one of the anticoagulants described in section 2.2.

1.31. Determination of the suitability of the donor shall be theresponsibility of a licensed physician and shall be. made by him orunder his supervision with the assistance of the necessary trainedattendants. Only those persons may serve as a source of blood who are inphysical condition to give blood, whose temperature and blood pressureare normal and who are freeof disease'transmissible by blood products asfar as can be determined from the donors history and from such physicalexamination and clinical tests as appear necessary for each donor on theday the blood is obtained.

4. Method of processing 4.1 Plasma acceptable for processing-Normalserum albumin shall be processed only from plasma containing not morethan 100 mg. hemoglobin per 100 ml. and prepared as indicated in section3, or from Normal Human Plasma prepared in accordance with the MinimumRequirements for that product.

4.2. Sepafation of the albumin fraction-Only those methods of processingwhich have been demonstrated to be capable of producing a final productfree of the agent(s) of viral hepatitis will be considered satisfactory.In addition, the processing method shall yield a product which will besafe and suitable for intravenous injection. The various steps inprocessing shall be conducted in as aseptic a manner as possible, andevery precaution of technique and cleanliness of equipment must beobserved to prevent unnecessary contamination either with bacteria orother deleterious matter-particularly pyrogenic material.

4.52. Accepted stabilizers for Normal Serum Albumin (Human) are:

(11) 0.04 M. sodium. acetyltryptophanate (b) 0.02 M. sodiumacetyltryptophanate and 0.02 M.

sodium caprylate 4.53. The pH of the final product is 6.9:L-0.4 asmeasured in a solution diluted to 1 percent in 0.15 M. NaCl.

4.54. The solution shall be rendered bacteriologically sterile byfiltration through a bacteria-excluding filter promptly after dissolvingin the diluent.

4.7. Heat treatment.Regardless of whether the final product is liquid ordried the albumin is heated during processing as a 25 percent solutionat an attained temperature of 60 C.-* -0.5 C. for not less than 10hours.

4.8. Inspection of final containers of liquid albumin followinglzeating.-All final containers of liquid albumin shall be held for notless than 7 days at 18-35 C. At the end of this incubation period thecontainers are inspected and any showing visible evidence of bacterialgrowth shall be discarded.

5. Control tests on the product 5.1. Sterility test.

5.2. Stability test-The turbidity is measured by comparison of the lightof wavelength 5461 A. (from a General Electric H4 mercury vapor lampthrough a Corning No. 3486 filter) scattered at an angle of 45 and thattransmitted at 0. One unit of the Mueller nephelometer as currently usedcorresponds to 0.0010 cm. absolute turbidity. The albumin shall meet thefollowing criteria:

(a) The turbidity of the 25 percent albumin solution after heattreatment must not be more than 16 units (preferably not more than 15units). It must not increase more than 5 units during 12 days at 50 C.(preferably notmore than 3 units).

(b) The turbidity must not increase more than 21 units (preferably notmore than 20 units) when the sample is heated for 50 hours at 57 C.

(0) The solution shall be substantially free from visible particles,both before heating and after 12 days at 50 C.

5.3. Concentration of pr0tein.--The amount of protein present shall bedetermined by a Kjeldahl N method on the basis of total N X 6.25(corrected for the Kjeldahl nitrogen content of stabilizer) and thecomposition of the proteins shall be determined by electrophoreticanalysis.

5.4. Albumin and globulin content.The total proteins in the finalproduct shall consist of not more than 3 percent of globulins asestimated electrophoretically in barbiturate buffer of ionic strength0.1 and pH 8.6.

5.5. Safety test.--A safety test shall be made on the contents of afinal container selected at random from each filling of. a lot. Theparenteral injection of 0.5 ml. into each of. 2 mice Weighingapproximately 20 grams and 5.0

ml. into each of 2 guinea pigs weighing approximately 350 grams shallcause neither significant symptoms nor death within 7 days.

5.6. Pyrogen test.A pyrogen test is made on thecontents of a finalcontainer selected at random from each filling of a lot. The test isperformed as described in the Memorandum of Details, except that theproduct is acceptable'if the temperature rise is less than 1.1 C. (20F.) on each of three hourly readings in each of three rab- 5 bitsfollowing the intravenous injection of 3.0 ml. per kilo of rabbitweight. The test provides for a retest.

5 .9. Sodium content.The final preparation must contain not more than0.014 gm. of sodium per gram of albumin (preferably not more than 0.013'gm.).

5.10. H emte cntent.The optical extinction at 403 mh. of a 1% albuminsolution in a 1 cm. cell it... m)

shall not exceed 0.25, which corresponds approximately to 4.6 mg. hemeper 25 grams of albumin, with the understanding that this limit will bereduced as soon as improved methods of blood collection and processingmake this possible.

These proteins may be used in a 3 to 4 percent solution. The totalamount of the protein solution used for treating the filter should be ina certain relation to the filter area; it has been found thatsatisfactory results are obtained when such an amount is used that 50-55mg. protein per square centimeter of the filter pad are available.

After having treated the pore filter with the human protein solution inorder to saturate the active inner surfaces of the pores, the proteinmaterial not tightly adsorbed must be cautiously removed. For thispurpose the filter is washed with sterile saline or with sterile Hankssolution until the last washing shows not more than traces of protein.While the presence of traces of human proteins in the filtrate isunobjectiona'ble it must be considered that proteins present in higherconcentrations have disturbing effects in the subsequent processing ofthe virus suspensions by interaction with inactivating agents or in theadministering of the vaccines by inducing allergic reactions invaccinees.

With the aid of the process according to the invention it is possible toretain the antigenicity of virus fiuids subjected to filtration on thehigh origin level before the filtration step.

The process according to the invention is applicable of virus of severalkinds. The process has been adopted with particular success for thetreating of poliomyelitis virus of the types I, II and III.

Filter materials which have been found to be most suitable for theprocess according to the invention are the cellulose type filter and inparticular the acetyl cellulose type filter. The mean pore dimensions ofthe filter pads should be in certain relation to the mean particlediameters of the virus e.g. in a relation of 3:1 to 10:1.

A filtration set suitable for using in the process according to theinvention is shown in the accompanying drawing which represents asectional view. y

The filtration set comprises a pressure tight container 1 having a screwcap 2 with an inlet 3 for compressed air. In the bottom of the containeran outlet 4 is pro vided connected with the tube '5 leading to thefilter drum 6. The filter drum is formed by two concave bottom-s 7 and 8each having a fitting 9 and 10 for the connection with the tube forsupplying the fiuid to be filtrated and the tube 11 for removing thefiltrate. The filter elements consisting of a stainless steel rack 12, apaper filter pad 14 (for instance Seitz No. 68) and an acetyl cellulosetype filter 13 are tightened between the edges of the bottoms 7 and 8 bymeans of the tightening elements 15.

The invention is illustrated in greater detail by the followingexamples:

Example 1 A bacteria tight filter pad of the acetyl cellulose typehaving a mean pore diameter of about 1000 Angstrom units was introducedinto the pressure filtration set as shown in the drawing. The filter pad(diameter 300 mm.) was boiled in distilled water during 30 minutes. Thenthe pad was placed on a paper filter of the Seitz type (No. 68) and bothwere put on the stainless steel rack of the filter set. The upper andlower bottoms of the drum were pressure tight closed. The'filtration setwas steam sterilized before use.

The filtration set was connected by sterilized tubing under sterileconditions to a container having a volume of 20 litres. A solutioncontaining a human plasma protein fraction (P.P.F. protein producedaccording-to the provisional Minimum Requirements of the NationalInstitutes of Health and thus a product derived from human bloodconsisting of not less than $2.7 albumin and not more than 15:globulins'other than gamma globulin by moving boundary electrophoresis)in a concentration of 3.5 weight/volume percent was subjected to heattreatment during a period of 10 hours at C. The turbidity of the heattreated solution was not higher than 30 Mueller units; the solution wasclarified by filtration; As per one square centimeter of acetylcellulose pad not more than 1.3 millilitres of the solution arenecessary, a total amount of 910 millilitres of the 3.5 percent P.-P.F.-solution are necessary to treat the filter pad. This volume was passedunder pressure through the acetyl cellulose pad. Then 15 litres of Hankssolution (21.5 millilitres per square centimeter) were passed throughthe filter in order to remove all loosely adsorbed protein material. Thelast filtrate of the washing. was tested with trichloro acetic acid todetermine absence of protein. If more than traces of protein weredetectable, additional washing with Hanks solution was necessary. Thefiltration set was disconnected from the 20 litre container and attachedto a 50 litre container for filtering virus suspension through theprepared filter. In three consecutive operations 50 litres each of virussuspensions of poliomyelitis of type I (strain Mahoney), type II (strainMEF-l), .and type III (strain :Saukett) were clarified by filtrationthrough Seitz type filter pads No. 68 and then passed through theprotein saturated acetyl cellulose filter pad under a pressure of 1.5atmospheres. Samples were taken before and after the filtration fortitration of the virus. A comparison of the two samples according to thetissue culture technique, the values being given in logs of 10indicating the dilution at which 5.0 percent of the tissue culturesshowed a cytopathogenic efrec-t, gave the tollowing results:

It is seen that in none of the three virus'suspensions significantlosses of antigenicity could be detected.

Example 2 The filtration set was prepared as described in Example 1. Asolution containing 4 weight/volume percent of human albumin (producedaccording to the Minimum Requirements of the National Institutes ofHealth for Human Albumin and thus a sterile preparation of the serumalbumin component of human blood comprising the final product, thetot-a1 protein content of which consists of not less than 96% albumin)was used for thepretreatment of the acetyl-cellulose filter pad whichsolution had been previously subjected to a heat'treatment during aperiod of 10 hours at a temperature of 60 C.

The heat treated solution was clarified by filtration. 1.3

millilitres per square centimeter of the acetyl cellulose filter neededa total amount of 910 millilitres of the-4 percent human albuminsolution. This volume was passed through the acetyl cellulose filter inorder to saturate the inner surfaces of the filter pores. Then thefilter was washed with Hanks solution (21.5 millilitres per squarecentimeter). The last washing gave a negative result with trichloroacetic acid. The filter prepared in this way was disconnected from thewashing container and connected to a sterilized container filled with 50litres of a poliomyelitis virus suspension of type I (strain Mahoney)which had beenpreviously clarified by filtration through Seitz typepaper filter pads No. 68. A-sample was taken to verify the initial titreof the virus suspension. Then the filtration of the virus suspensionthrough the albumin saturated acetyl cellulosefilter was performed undera pressure of 1.5 atmospheres. A second sample was taken from thefiltrate to determine the final titre. A comparision of the titre valuesgave the following results: Initial titre: 6.9 per 0.25 ml. of the virussuspension; final titre: 6.8 ,per 0.25 ml.; a parallel experiment gavethe :respective values of 6.8 and 6.5 (titre values are expressed inlogs of 10 giving the dilution which shows a -50 percent cytopathogeniceffect in the tissue culture titration technique).

Example 3 A filtration set was prepared as described in Example 1, butno treatment with a protein solution was carried out. A virus suspensionof poliomyelitis .virus type I (strainMahoney) was clarified byfiltration. The initial titre was determined and then 50 litres of thesuspension were filtered through the acetyl cellulose filter. Againasample was taken for determination of the final titre. The respectivetitres (double estimations) of the prefiltrationsample was: 6.8 and 6.9,while the titre after the filtration was: 3.0 and 2.0 per 0.25millilitre of suspension. This ditference makes obvious the importantloss of antigenicity during filtration according to the common practice.

The acetyl cellulose filter used in the procedures according to theExamples 1 to 3 may be prepared in the following way:

A cellulose card board in a thickness of 0.5 mm. is immersed in asolution of 4 percent by weight acetyl cellulose in glacial acetic acidduring a period of 1 hour. Then the treated card board is put intoflowing water during a period of 24 hours. After this washing the cardboard is dried. It has a mean pore diameter of about 800 to 1200Angstrom units.

What we claim is:

1. A method for eliminating the antigen-adsorption activity of a porefilter of the acetyl cellulose type having a mean pore diameter of about800 to 1200 angstrom units and destined for the filtration of virusfluids comprising treating the pore filter with a human protein solutionselected from the group consisting of plasma protein fraction comprisinga product derived from human blood consisting of not less than 851 2.7albumin and not more than 15:2.7 globulinsother than gamma globulin bymoving-boundary electrophoresis and a sterile preparation of the serumalbumin component of human'blood, the protein content of which consistsof not less than 96% albumin, in a concentration of 3% to 4%, saidsolution being heat treated for a period of not less than 10 hours at atemperature of 60 C., to saturate the adsorptive capacity of the innerpore surfaces of the filter with proteins of human origin, and removingall protein material not tightly absorbed to the inner pore surfaces ofthe filter 'by washing 'it with a protein-free physiological salinesolution.

2. A method for eliminating the adsorptive activity for antigen of apore filter of theacetyl cellulose type having a mean pore diameter ofabout 800 to 1200 angstrom units and destined for the filtration ofvirus fluids, comprising treating said pore filter with a proteinsolution selected from the group consisting of plasma protein fractioncomprising a product derived from human blood consisting of not lessthan 85:27 albumin and not more than 15:2.7 globulins other than gammaglobulin by moving boundary electrophoresis and a sterile preparation ofthe serum albumin component of human blood, the protein content of whichconsists of not less than 5 96% albumin, the solution containingproteins of human origin in the range of the electrophoretic mobilityclasses between 8.0 and --3.0 cm. voltsec.- in a concentration of 3 to 4percent, being treated for a period of not less than 10 hours at atemperature of 60 C. and

10 free from denaturated protein fractions, and having a turbidity ofnot more than 30 Mueller units, and then removing all protein materialnot tightly adsorbed to the inner pore surfaces of the filter by washingit with sterile Hanks solution.

3. A method as set forth in claim 2 in which such an amount of theprotein solution is used for treating the pore filter that 50 to 55 mg.protein are available per square centimeter of the filter.

4. A method for the filtration of virus fluids having a givenantigenicity comprising treating a pore filter of the acetyl cellulosetype having a mean pore diameter of about 800 to 1200 angstrom units,being three to four times greater than the mean particle diameter of thevirus to be filtrated, comprising pretreating said pore filter with aheat-inactiviated human protein solution selected from the groupconsisting of plasma protein fraction comprising a' product derived fromhuman blood consisting of not lessthan 85:2.7 albumin and not more than15 -2.7 globulins other than gamma globulin by moving 3O boundaryelectrophoresis and a sterile preparation of the serum albumin componentof human blood, the protein contentof which consists of not less than96% albumin, the solution having a concentration of 3 to 4 percent andbeing free from denaturated protein fractions, in order to saturate theadsorptive capacity of the filter pads with proteins of human origin,washing the pore filter with a proteinfree physiological saline solutionto remove all protein materials not tightly adsorbed to the innersurface of the pores, and passing the virus fluid through the filter,

40 the filtrate containing all of antigenicity of the origin virusfluid.

5. A method as set forth in claim 4 in which fluids containing virusselected from poliomyelitis virus of the type I, 11 and III are treated.

References Cited in the file of this patent FOREIGN PATENTS 1,012,436Germany July 18, 1957 5 OTHER REFERENCES Krueger et al.: J our. Gen.Physiol., vol. 13, pp. 409-419 (1930).

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C. Thomas Co., Springfield, Ill. (1948), pp. 429-436.

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1. A METHOD FOR ELIMINATING THE ANTIGEN-ADSORPTION ATIVITY OF A POREFILTER OF THE ACETYL CELLULOSE TYPE HAVING A MEAN PORE DIAMETER OF ABOUT800 TO 1200 ANGSTROM UNITS AND DESTINED FOR THE FILTRATION OF VIRUSFLUIDS COMPPRISING TREATING THE PORE FILTER WITH A HUMAN PROTEINSOLUTION SELECTEF FROM THE GROUP CONSISTING A PLASMA PROTEIN FRACTIONCOMPRISING A PRODUCT DERIVSED FROM HUMAN BLOOD CONSISTING OF NOT LESSTHAN 85+2.7 ALBUMIN AND NOT MORE THAN 15-2.7 GLOBULINS OTHER THAN GAMMAGLOBULIN BY MOVING BOUNDARY ELECTROPHORESIS AND A STERILE PREPARA-TIONOF THE SERUM ALBUMIN COMPONENT OF HUMAN BLOOD, THE PROTEIN CONTENT OFWHICH CONSISTS OF NOT LESS THAN 96% ALBUMIN, IN A CONCENTRSATION OF 3%TO4%, SAID SOLUTIONN BEING HEAT TREATED FOR A PERIOD OF NOT LESS THAN 10HOURS AT A TEMPRATURE OF 60* C ., TO SATURATE THE ADSORPTIVE CAPACITY OFTHE INNER PORE SURFACES OF THE FILTER WITH PROTEINS OF HUMAN ORIGIN, ANDREMOVING ALL PROTEIN MATERIAL BY WASHING IT WITH A PROTEIN-FREEPHYSIOLOGICAL SALINE SOLUTION.